CN113736897B - Primer group, kit and method for detecting vibrio parahaemolyticus and vibrio cholerae based on dual RAA-LFD technology - Google Patents
Primer group, kit and method for detecting vibrio parahaemolyticus and vibrio cholerae based on dual RAA-LFD technology Download PDFInfo
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Abstract
The invention discloses a specific primer combination, a kit and a method for simultaneously detecting vibrio parahaemolyticus and vibrio cholerae based on a dual RAA-LFD technology, belonging to the field of rapid detection of food-borne pathogenic bacteria. The primer has strong specificity, and can accurately detect the vibrio parahaemolyticus and the vibrio cholerae from other vibrio and other pathogenic bacteria; the sensitivity is high, the genome sensitivity reaches 1fg, and the sensitivity of pure bacterial solutions is respectively 10 4 CFU/mL and 10 3 CFU/mL; but also reduces the influence of dimers; the reaction is carried out for 15min at a lower reaction temperature (37 ℃) and the test strip can finish the detection after detecting for 5min, thereby solving the problems of long detection period of vibrio parahaemolyticus and vibrio cholerae, high instrument and equipment dependence, high difficulty in detecting vibrio parahaemolyticus and vibrio cholerae at the same time and the like. Has low requirements on equipment and good application prospects in field detection of fields, aquaculture points and the like.
Description
Technical Field
The invention belongs to the technical detection field of molecular biology, and particularly relates to an improved primer, a kit and a detection method for simultaneously detecting vibrio parahaemolyticus and vibrio cholerae based on a dual RAA-LFD technology.
Background
Vibrio parahaemolyticus (Vibrio parahaemolyticus) and Vibrio cholerae (Vibrio cholerae) are major pathogenic bacteria in aquatic products. Vibrio parahaemolyticus is widely used in aquatic animals such as fish, shrimp and shellfish, and has been considered as an important cause of poisoning of global aquatic products since Fujino et al were first isolated from sardine in 1950. Vibrio cholerae has more than 200 serogroups, and O1 and O139 serovars can cause cholera. Vibrio cholerae is particularly prevalent in areas where conditions are poor or where development is relatively late. It is estimated that 300 to 500 tens of thousands of cases and more than 10 tens of thousands of deaths occur annually worldwide. The existence of vibrio parahaemolyticus and vibrio cholerae in the aquaculture environment can cause the symptoms of slow response, whitening of muscles, anorexia, gill yellowing and ulcer of aquatic animals such as shrimps and the like, has high mortality rate and brings economic loss to the aquaculture industry. Therefore, the rapid and accurate detection of the vibrio parahaemolyticus and the vibrio cholerae is a precondition for effectively controlling the popularity and the spread of the vibrio parahaemolyticus and the vibrio cholerae, can reduce the economic loss of the aquaculture industry and ensure the health of consumers.
The traditional culture method is a gold standard for detecting vibrio, but has long time consumption and large workload, and usually needs a week or more, and cannot meet the requirement of rapid detection of vibrio parahaemolyticus and vibrio cholerae in aquatic products. The polymerase chain reaction (Polymerase chain reaction, PCR) is applied to the detection of Vibrio parahaemolyticus and Vibrio cholerae due to the characteristics of high detection accuracy, high detection speed and the like. However, the PCR method requires the assistance of expensive thermal cycling equipment, which limits its use in field condition detection and basic laboratories. In addition, the loop-mediated isothermal amplification (Loop mediated isothermal amplification, LAMP) method has the characteristics of simple operation and high cost efficiency, has been developed for detecting vibrio parahaemolyticus and vibrio cholerae in an aquatic product culture environment, but the LAMP method needs to design 4-6 pairs of primers and is complex.
As a novel nucleic acid isothermal amplification technology, the recombinase isothermal amplification (Isothermal recombinase aided amplification, RAA) is a simple, rapid, specific, sensitive and economical molecular detection method for identifying various pathogenic bacteria. At present, a method (RAA-LFD) based on RAA and a lateral flow chromatography test strip (LFD) is successfully applied to rapid detection of food-borne pathogenic bacteria such as vibrio vulnificus, salmonella, staphylococcus aureus and the like, and field diagnosis in remote areas such as the field is realized. However, a dual RAA-LFD method has not been developed for simultaneous detection of Vibrio parahaemolyticus and Vibrio cholerae. The dual RAA-LFD method has important significance for the detection and epidemic control of vibrio parahaemolyticus and vibrio cholerae.
Disclosure of Invention
The invention aims to overcome the defects of the detection method in the prior art, and establishes a method for simultaneously detecting vibrio parahaemolyticus and vibrio cholerae based on a dual RAA-LFD technology so as to realize rapid, specific and accurate detection of vibrio parahaemolyticus and vibrio cholerae in environments with limited resources such as the wild.
The technical scheme of the invention is as follows:
a dual specificity primer combination for detecting vibrio parahaemolyticus and vibrio cholerae based on a dual RAA-LFD technology comprises two groups of primers; the first group of primers is used for specifically amplifying vibrio parahaemolyticus, the upstream primer sequence contains a nucleotide sequence shown as SEQ ID No.1, and the downstream primer sequence contains a nucleotide sequence shown as SEQ ID No.2;
the second set of primers is used for specifically amplifying vibrio cholerae, the upstream primer sequence contains a nucleotide sequence shown as SEQ ID No.3, and the downstream primer sequence contains a nucleotide sequence shown as SEQ ID No. 4.
Preferably, the first group of primers is used for specifically amplifying vibrio parahaemolyticus, the upstream primer sequence is shown as SEQ ID No.1, and the downstream primer sequence is shown as SEQ ID No.2;
the second group of primers is used for specifically amplifying vibrio cholerae, the upstream primer sequence is shown as SEQ ID No.3, and the downstream primer sequence is shown as SEQ ID No. 4.
SEQ ID No.1：tccaaaacgaggctatcaactcatttgcact
SEQ ID No.2：tcgctaaagacggctctacgattgtttctacc
SEQ ID No.3：ccattttcacataagatttctacctctggt
SEQ ID No.4：atgagtcgtcaagttttaaatcactcattc
Further, the first group of primers and the second group of primers, wherein the 5 'end of the upstream primer of one group is modified with a biotin compound, and the 5' end of the downstream primer is modified with a marker 1; the 5 'end of the other group of upstream primer modifies the marker 2, and the 5' end of the downstream primer modifies the biotin compound. The markers 1 and 2 are digoxin or fluorescein compounds.
In a preferred embodiment of the present invention, the upstream primer 5 'end of the first primer group is modified with a biotin compound, and the downstream primer 5' end is modified with a fluorescein compound; the 5 'end of the upstream primer of the second group of primers is modified with digoxin, and the 5' end of the downstream primer is modified with biotin compounds.
The biotin compound is biotin, and the fluorescein compound is 6-carboxyfluorescein (6-FAM) or Fluorescein Isothiocyanate (FITC), and more preferably 6-carboxyfluorescein.
The dual specific primer combination can be used for simultaneously detecting vibrio parahaemolyticus and vibrio cholerae.
The dual specificity primer combination can be used for preparing a kit for simultaneously detecting vibrio parahaemolyticus and vibrio cholerae.
A kit for detecting vibrio parahaemolyticus and vibrio cholerae based on a dual RAA-LFD technology contains the dual specific primer combination.
Preferably, the kit further comprises at least one of a RAA nucleic acid amplification kit and a lateral flow chromatography test strip.
The RAA nucleic acid amplification kit comprises freeze-dried powder, a basic buffer solution, magnesium acetate, purified water, negative quality control products and positive quality control products.
And a T1 detection line and a T2 detection line are arranged on the lateral flow chromatography test strip and are respectively used for capturing a marker 1 and a marker 2.
A method for simultaneously detecting vibrio parahaemolyticus and vibrio cholerae based on a dual RAA-LFD technology comprises the following steps:
(1) Mixing the specific primer combination with a sample to be detected, preparing an RAA reaction system, and carrying out amplification reaction for 10-30min at 36-38 ℃;
(2) And (3) dripping the amplified product to the tail end of a sample pad of the lateral flow chromatography test strip, vertically placing the sample pad into buffer solution, reacting for 5min, and reading the result.
In step (1), the reaction time is preferably 15 to 20 minutes, more preferably 15 minutes.
And a T1 detection line and a T2 detection line are arranged on the lateral flow chromatography test strip and are respectively used for capturing the marker 1 and the marker 2.
The T1 detection line, the T2 detection line and the quality control line of the lateral flow chromatography test strip are respectively marked with an antibody against the marker 1, an antibody against the marker 2 and a ligand of the biotin compound. Preferably, the anti-fluorescein antibody is marked on the T1 detection line of the lateral flow chromatography test strip, the anti-digoxin antibody is marked on the T2 detection line, and the biotin ligand is marked on the quality control line.
In the step (1), the total DNA of the sample to be detected is extracted. More preferably, the sample to be tested is enriched and total DNA is extracted.
The result is judged as follows:
(1) When the T1 detection line, the T2 detection line and the quality control line are all in strips, the sample is indicated to contain vibrio parahaemolyticus and vibrio cholerae simultaneously;
(2) When only the T1 detection line and the quality control line are in a strip, the sample contains vibrio parahaemolyticus and does not contain vibrio cholerae;
(3) When only the T2 detection line and the quality control line are in a strip, the sample contains vibrio cholerae and does not contain vibrio parahaemolyticus;
(4) When only the quality control line has stripes and the detection line has no stripes, the result is negative, namely the sample does not contain vibrio parahaemolyticus and vibrio cholerae, or the content of vibrio parahaemolyticus and vibrio cholerae is lower than the detection limit of the dual RAA-LFD method;
(5) And when the detection line and the quality control line are not provided with strips, the detection result is invalid.
According to the invention, primers are respectively designed according to a vibrio parahaemolyticus specific gene (toxR gene) and a vibrio cholerae specific gene (Ompw gene), and as a result, the false positive result appears when the double RAA-LFD detection is performed based on the primers due to the influence of primer dimers. Therefore, the obtained primer is subjected to base substitution to avoid the generation of dimer, and the primer with strong specificity and high sensitivity is obtained, so that vibrio parahaemolyticus and vibrio cholerae can be accurately detected from other vibrio and other pathogenic bacteria.
The invention has the beneficial effects that:
(1) The dual specificity primer based on the dual RAA-LFD technology provided by the invention has the advantages that after the sequence is improved, the phenomenon of false positive results caused by primer dimer generation can not occur, the specificity is strong, and the sensitivity is high;
(2) The kit based on the dual RAA-LFD technology provided by the invention can detect vibrio parahaemolyticus and vibrio cholerae simultaneously, and is simple and convenient to operate;
(3) The detection method for simultaneously detecting the vibrio parahaemolyticus and the vibrio cholerae based on the dual RAA-LFD technology provided by the invention has good characteristics, and can accurately detect the vibrio parahaemolyticus and the vibrio cholerae in other vibrio and pathogenic bacteria, and the specificity reaches 100%;
(4) The primer combination, the kit and the method have high sensitivity, the detection limit of the genome DNA of the vibrio parahaemolyticus and the vibrio cholerae reaches 1fg, and the detection limit of the pure bacterial liquid of the vibrio parahaemolyticus and the vibrio cholerae reaches 10 respectively 4 CFU/mL and 10 3 CFU/mL; when enriched, as low as 10 can be detected 0 CFU/g of Vibrio parahaemolyticus and Vibrio cholerae;
(5) The detection method for simultaneously detecting the vibrio parahaemolyticus and the vibrio cholerae based on the dual RAA-LFD technology has low requirements on instruments and equipment, can complete amplification after incubation for 15min at a lower reaction temperature of 37 ℃, and can complete detection after detection for 5min by using a test strip. Meanwhile, after the amplification products are detected by LFD, the results are available to naked eyes, the result judgment is simple, and the method is suitable for rapidly detecting vibrio parahaemolyticus and vibrio cholerae in environments with limited resources such as the wild and has good application prospect.
Drawings
FIG. 1 is a software designed primer of example 1 wherein 1: toxR-F/R: ompw-F/r=1.25 μm: 2.5. Mu.M; NT: negative control.
FIG. 2 is a diagram showing the results of a primer combination screening by the dual RAA-LFD method according to the present invention, wherein 1: M-toxR-F/R: ompw-F/r=1.25 μm: 2.5. Mu.M; 2: toxR-F/R: M-Ompw-F/r=1.25 μm: 2.5. Mu.M; 3: M-toxR-F/R: M-Ompw-F/r=1.25 μm: 2.5. Mu.M; NT: negative control.
FIG. 3 is a graph of the optimized reaction time obtained by the dual RAA-LFD method of example 3, wherein 1-5 represent reaction times of 10min,15min,20min,25min, and 30min, respectively; NT is a negative control.
FIG. 4 is a graph showing the specificity of the dual RAA-LFD method of the present invention, wherein 1: vibrio cholerae GIM1.449+ Vibrio parahaemolyticus ATCC17802;2: vibrio parahaemolyticus ATCC17802;3: vibrio parahaemolyticus SH06;4: vibrio cholerae GIM1.449;5: vibrio cholerae CICC23794;6-13: vibrio fluvialis JS-X-S-4-2, vibrio mimicus CM-X-W-2-1, vibrio mediterraneans JS-X-5-2-3, staphylococcus aureus ATCC29213, listeria monocytogenes ATCC19115, salmonella enteritidis SAL4, klebsiella pneumoniae BS-X-S-1; pseudomonas aeruginosa; NT is a negative control.
FIG. 5 is a graph showing the detection sensitivity results of the dual RAA-LFD method of the present invention, wherein A is a graph showing the genomic sensitivity results; 1-7 represent the DNA concentrations of Vibrio parahaemolyticus and Vibrio cholerae 1ng,100pg,10pg,1pg,100fg,10fg,1fg, NT-negative control, respectively; b is a pure bacterial liquid sensitivity result graph; 1-8 respectively represent the concentration 10 of pure bacterial liquid of vibrio parahaemolyticus and vibrio cholerae 7 CFU/ml-10 0 CFU/ml, NT-negative control.
FIG. 6 is a graph showing the results of a simulation of a sample by the dual RAA-LFD method according to the present invention, wherein A represents an enrichment time of 0h; b represents enrichment time of 2h; c represents enrichment time of 4 hours; d represents enrichment time of 6 hours; 1-8 represent Vibrio parahaemolyticus and Vibrio cholerae pollution amount 10, respectively 7 CFU/g～10 0 CFU/g, NT-negative control.
Detailed Description
The technical solutions in the present application will be further described and explained with reference to specific embodiments and drawings. It is apparent that the specific embodiments described herein are only some, not all, of the embodiments of the present application. Modifications and substitutions of detail and form of the technical solution of the present invention may be made by those skilled in the art without creative efforts, and these modifications and substitutions are all within the scope of the present invention.
Unless otherwise indicated, the raw materials and chemical reagents in the examples are conventional commercial products, and the technical means are conventional means used by those skilled in the art.
EXAMPLE 1 design of Dual RAA-LFD reaction primers
The sequences of specific genes (toxR gene) of Vibrio parahaemolyticus and specific genes (Ompw gene) of Vibrio cholerae were downloaded from GenBank as targets for Primer design, their conserved sequences were searched for by DNAMAN, primers were designed by Primer Premier 5 according to RAA Primer design principle, biotin (Biotin) and carboxyfluorescein (6-FAM) were labeled at the 5 'ends of primers toxR-F and toxR-R, respectively, digoxin (DIG) and Biotin (Biotin) were labeled at the 5' ends of primers Ompw-F and Ompw-R. Primers were synthesized by the company limited by the biological engineering (Shanghai) and the primer sequences are shown in the following table:
and (3) detecting by a double RAA-LFD method, and finding that false positive phenomenon occurs.
The RAA reaction was slightly modified according to the protocol (RAA nucleic acid amplification kit, jiangsu Qian). The reaction system (50. Mu.L) was as follows:
25. Mu.L of buffer, 1.25. Mu.M toxR-F1. Mu.L, 1.25. Mu.M toxR-R1. Mu.L, 2.5. Mu.M Ompw-F1. Mu.L, 2.5. Mu.M Ompw-R1. Mu.L, and 16.5. Mu.L of purified water. After fully mixing, subpackaging 45.5 mu L of the mixture into freeze-dried powder, transferring the liquid into a 1.5mL centrifuge tube after fully dissolving and thawing the dry powder, then respectively adding 1 mu L of total DNA templates of vibrio parahaemolyticus and vibrio cholerae, and finally dropwise adding 2.5 mu L of magnesium acetate solution on each centrifuge tube cover, slightly oscillating and centrifuging. And (3) placing the reaction tube into a water bath kettle to react for 15min at 37 ℃, taking 10 mu L of amplification product, dripping the amplification product onto a sample pad of the lateral flow chromatography test strip, vertically placing the tail end of the sample pad of the lateral flow chromatography test strip into a centrifuge tube added with 100 mu L of buffer solution, reacting for 5min, and reading the result.
As shown in FIG. 1, the test strip is sequentially provided with a T1 detection line (Vibrio parahaemolyticus), a T2 detection line (Vibrio cholerae) and a quality control line (C) from bottom to top. After amplification using primers (toxR-F/R and Ompw-F/R) bands (false positive results), especially T2, appear on both T lines of the negative control.
It is presumed that the formation of primer dimer at the 3' -end affects the detection result and false positive occurs. When the primer toxR-F and the primer toxR-R form a primer dimer (carrying Biotin and 6-FAM) with the primer Ompw-R, a strip appears at the detection line of the test strip T1, and the judgment of the result is affected; similarly, when the primer Ompw-F and the primer Ompw-R and the primer toxR-F form a primer dimer (carrying Biotin and DIG) with the primer Ompw-F, a band appears at the detection line of the test strip T2, which affects the judgment of the result. Thus, to reduce interference of primer dimer in RAA amplification system, the primers toxR-F/R and Ompw-F/R were modified.
Analyzing 3' -end primer dimers formed by the primer toxR-F and the primer toxR-R, toxR-R and the primer Ompw-R, the primer Ompw-F and the primer Ompw-R and the primer toxR-F and the primer Ompw-F, and carrying out primer base substitution according to the analysis result so as to reduce the possibility of generating the primer dimers.
The primers toxR-F and Ompw-R were modified, respectively. Namely, the 28 th base "t" in the primer toxR-F is replaced with "c", and the 28 th base "a" in the primer Ompw-R is replaced with "t". After one base in the primer toxR-F and the primer Ompw-R is replaced, the generation of 3' -end primer dimer is reduced. The modified primer sequences are shown in the following table:
example 2 screening of double RAA-LFD reaction primer combinations
To verify the amplification efficiency of the modified primers, the unmodified primers and the modified primers were combined two by two, and the optimal primer combination was selected by performing a double RAA-LFD method. The combination is respectively as follows: toxR-F/R and Ompw-F/R, M-toxR-F/R and Ompw-F/R, toxR-F/R and M-Ompw-F/R, M-toxR-F/R and M-Ompw-F/R.
The RAA reaction was carried out as described in example 1.
As a result, as shown in FIG. 2, the test strips showed correct positive signals after amplification using the modified primers (toxR-F/R and M-Ompw-F/R, M-toxR-F/R and M-Ompw-F/R) (negative control amplification was normal and did not affect the positive amplification results). However, the difference in brightness between the two T lines after amplification using the primers M-toxR-F/R and M-Ompw-F/R was large. Therefore, the primers toxR-F/R and M-Ompw-F/R were selected for subsequent experiments.
As can be seen, the substitution of the 28 th base "t" in primer toxR-F with "c" and the 28 th base "a" in primer Ompw-R with "t" reduces the possibility of producing 3' terminal primer dimer.
Example 3 optimization of Dual RAA-LFD reaction time
The system was formulated as in example 2 and the amplification time of the RAA was optimized.
The reaction tube is respectively incubated for 10min,15min,20min,25min and 30min at 37 ℃ and then 10 mu L of amplified products are taken for test strip detection. As a result, as shown in FIG. 3, the brightness of the test strip on the two detection lines was changed with the increase of the reaction time. As the reaction time increases, the brightness of the strip on the T1 detection line gradually increases, no strip appears on the T1 detection line when the reaction time is 10min, and the brightness of the strip on the T1 detection line reaches stability when the reaction time is 20min. In contrast, the brightness of the band on the T2 detection line decreases with increasing reaction time, and the longer the reaction time, the darker the brightness of the band. Since the brightness of the bands on the T1 and T2 detection lines is equivalent after 15min of reaction, the optimal reaction time for the dual RAA-LFD method is 15min.
EXAMPLE 4 Dual RAA-LFD method specificity evaluation
Under the optimal reaction conditions, the dual RAA-LFD method is carried out on two strains of vibrio cholerae, two strains of vibrio parahaemolyticus and 8 strains of other food-borne pathogens (see the following table) so as to evaluate the specificity of the dual RAA-LFD method.
The specific evaluation result of the dual RAA-LFD is shown in figure 4, only vibrio parahaemolyticus and vibrio cholerae generate positive strips, and other 8 pathogenic bacteria only generate quality control lines, which shows that the method can be used for detecting vibrio parahaemolyticus and vibrio cholerae simultaneously, has no cross reaction with other pathogenic bacteria, and has good specificity.
Example 5 Dual RAA-LFD sensitivity assessment
(1) Genome sensitivity evaluation
Ten times serial dilutions were made on the genomic DNA of vibrio cholerae and vibrio parahaemolyticus, 1 μl of each DNA was taken and subjected to double RAA-LFD detection under optimal RAA reaction conditions to determine the detection limit.
As shown in FIG. 5-A, the genomic sensitivity of the dual RAA-LFD method gradually decreases with decreasing DNA concentration, while the intensity of the band on the T1 detection line gradually decreases, whereas the intensity of the band on the T2 detection line shows a tendency of increasing and decreasing, which is probably due to competition of the two primers in the dual RAA reaction system. However, the bands were seen on both detection lines when the DNA concentration was as low as 1fg, so that the genome detection limit for detecting Vibrio parahaemolyticus and Vibrio cholerae by the dual RAA-LFD method was 1fg.
(2) Pure bacterial liquid sensitivity evaluation
Ten times serial dilution of pure bacteria liquid of cholera vibrio and parahaemolyticus vibrio is carried out by using physiological saline to obtain 10 7 CFU/mL-10 0 The CFU/mL pure bacterial liquid is taken, 1mL pure bacterial liquid with different concentrations is put into a 1.5mL centrifuge tube, and DNA is extracted by a boiling method. The extracted DNA is used for double RAA-LFD detection.
The sensitivity of pure bacterial liquid by the dual RAA-LFD method is shown in figure 5-B, the brightness of the strip on the T1 detection line gradually decreases along with the decrease of the bacterial liquid concentration, and when the concentration of the pure bacterial liquid of the vibrio parahaemolyticus is 10 4 The band can be observed on the T1 detection line at CFU/mL, so the detection limit of the dual RAA-LFD method for detecting the pure bacterial liquid of the vibrio parahaemolyticus is 10 4 CFU/mL; the brightness of the strip on the T2 detection line shows a trend of increasing and decreasing along with the decrease of the bacterial liquid concentration, and when the concentration of the pure bacterial liquid of the vibrio cholerae decreases to 10 2 CFU/mLThe T2 detection line has no visible band, so the detection limit of the dual RAA-LFD method for detecting the pure bacterial liquid of the vibrio cholerae is 10 3 CFU/mL。
Example 5 simulation sample detection
Vibrio cholerae GIM1.449 and Vibrio parahaemolyticus ATCC17802 were streaked on TCBS plates, incubated overnight at 37℃and then inoculated in 5mL of alkaline peptone water containing 3% sodium chloride, followed by incubation at 37℃for 6 hours.
5g of shrimp meat was weighed in a sterile petri dish, and 100. Mu.L of Vibrio parahaemolyticus and 200. Mu.L of Vibrio cholerae were added dropwise to the shrimp surface to achieve 10 0 CFU/g～10 7 Pollution amount of CFU/g. Shrimp meat contaminated with different concentrations of vibrio parahaemolyticus and vibrio cholerae is transferred into a sterile homogenizing bag filled with 45mL of 2% APW, and after being beaten for 2min by a beater, 5mL is taken out of a test tube, and after 0, 2, 4 and 6h are respectively enriched at 37 ℃/200rpm, 1mL of filtrate is taken out for extracting DNA by a boiling method. And detecting the obtained DNA by a double RAA-LFD method.
The results of the simulated sample testing are shown in FIG. 6. When not enriched, the dual RAA-LFD method can detect that the shrimp meat is polluted by 10 percent 5 CFU/g of Vibrio parahaemolyticus and Vibrio cholerae; after 2h of enrichment, the dual RAA-LFD method can detect that the shrimp meat is polluted by 10 percent 2 CFU/g of Vibrio parahaemolyticus and Vibrio cholerae; the pollution in shrimp meat is as low as 10 0 CFU/g of Vibrio parahaemolyticus was enriched for 4 hours and detected by the dual RAA-LFD method, and contained 10 0 The shrimp meat of CFU/g Vibrio cholerae can be detected by the dual RAA-LFD method after 6 hours of enrichment.
The above examples illustrate that the amplification primers for simultaneously detecting Vibrio parahaemolyticus and Vibrio cholerae based on the dual RAA-LFD technology have strong specificity and high sensitivity; the base modification of the amplification primer reduces the interference of primer dimer to the experiment, and does not affect the amplification specificity of RAA; the method is reacted for 15min at the body temperature (37 ℃), the simultaneous detection of the vibrio parahaemolyticus and the vibrio cholerae can be realized by detecting the test strip for 5min, the detection speed is high, the equipment requirement is low, and the method has good application prospect in field detection and the like.
Sequence listing
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Claims (5)
1. A specific primer combination for detecting vibrio parahaemolyticus and vibrio cholerae based on a dual RAA-LFD technology is characterized by comprising two groups of primers; the first group of primers is used for specifically amplifying vibrio parahaemolyticus, the upstream primer sequence is shown as SEQ ID No.1, and the downstream primer sequence is shown as SEQ ID No.2;
the second group of primers is used for specifically amplifying vibrio cholerae, the upstream primer sequence is shown as SEQ ID No.3, and the downstream primer sequence is shown as SEQ ID No. 4; the 5 'end of the upstream primer of the first group of primers is modified with biotin, and the 5' end of the downstream primer is modified with 6-carboxyfluorescein; the 5 '-end of the upstream primer of the second primer group is modified with digoxin, and the 5' -end of the downstream primer is modified with biotin.
2. A specific primer combination according to claim 1 for use in preparing a kit for simultaneous detection of Vibrio parahaemolyticus and Vibrio cholerae, or for non-diagnostic purposes.
3. A kit for detecting vibrio parahaemolyticus and vibrio cholerae based on a dual RAA-LFD technology is characterized by comprising the dual specific primer combination of claim 1, and further comprising a RAA nucleic acid amplification kit and a lateral flow chromatography test strip.
4. A method for detecting vibrio parahaemolyticus and vibrio cholerae based on a double RAA-LFD technology for non-diagnostic purpose is characterized by comprising the following steps:
(1) Mixing the dual specific primer combination of claim 1 with a sample to be tested, preparing a RAA reaction system, and performing amplification reaction for 10-30min at 36-38 ℃;
(2) Dripping the amplified product to the tail end of a sample pad of the lateral flow chromatography test strip, vertically placing the sample pad into buffer solution, reacting for 5min, and reading the result; the lateral flow chromatography test strip is provided with a T1 detection line and a T2 detection line which are respectively used for capturing 6-carboxyfluorescein and ground Xin Gao.
5. The method according to claim 4, wherein in the step (1), the reaction time is 15 to 20 minutes.
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